Hemophilia is an inherited disease which has been known for centuries, but it is only within the last few decades that it has been possible to differentiate among the various forms; hemophilia A and hemophilia B. Hemophilia A is caused by strongly decreased level or absence of biologically active coagulation factor VIII, which is a protein normally present in plasma.
Factor VIII (FVIII) is a large and complex glycoprotein that participates in the blood coagulation cascade. Deficiencies in FVIII production in vivo caused by genetic mutation can cause hemophilia, which is treated by infusion of purified preparations of human FVIII (Lee, Thromb Haemost, 82:516-524, 1999). The first purified FVIII products were derived from human serum, isolated from the cryoprecipitate from the Cohn fractionation process.
Research efforts have focused on the development of methods for creating and isolating highly purified, biologically active factor VIII in full-length and derivative forms. Advantages of a highly purified protein include reduced levels of extraneous proteins in the therapeutic mix as well as a decreased likelihood of the presence of infectious agents. A more purified form of factor VIII may also be administered in smaller doses, possibly reducing the risk of developing anti-factor VIII antibodies, as lower doses would be less challenging to the immune system.
Significant steps have been taken toward the recombinant production of factor VIII beginning with the isolation of biologically active factor VIII fragments. See, U.S. Pat. No. 4,749,780; U.S. Pat. No. 4,877,614. The gene encoding the full-length human factor VIII protein was isolated by taking advantage of its sequence homology with porcine factor VIII. See, U.S. Pat. No. 4,757,006. DNA sequences encoding the human coagulation cofactor, Factor VIII:C (FVIII), are also known in the art [see, e.g., Toole et al., Nature 312:312-317, 1984; Wood et al., Nature 312:330-337, 1984; Vehar et al.; Nature 312:337-342, 1984], as well as methods for expressing them to produce recombinant FVIII [see e.g. Toole, U.S. Pat. No. 4,757,006; WO 87/04187, WO 88/08035 and WO 88/03558]. The expression of human and porcine protein having factor VIII:C procoagulant activity was also described in U.S. Pat. No. 4,575,006. Active variants and analogs of FVIII protein, and DNA sequences encoding them, have also been reported [see, e.g. Toole, U.S. Pat. No. 4,868,112; EP 0 786 474; WO 86/06101 and WO 87/07144]. Generally, such variants and analogs are modified such that part or all of the B domain is missing and/or specific amino acid positions are modified, for example, such that normally protease-labile sites are resistant to proteolysis, e.g., by thrombin or activated Protein C. Other analogs include modification at one or more lysine and/or tyrosine residues.
Since severe side effects involving the production of anti-factor VIII antibodies exist with the administration of the protein isolated from both human and non-human sources, truncated lower molecular weight proteins exhibiting procoagulant activity have been designed. U.S. Pat. No. 4,868,112 reports an alternative method of treatment with lower molecular weight porcine factor VIII of approximately 2000 amino acids exhibiting similar procoagulant activity as full-length factor VIII. The removal of certain amino acids and up to 19 of the 25 possible glycosylation sites reduced the antigenicity of the protein and thereby the likelihood of developing anti-factor VIII antibodies. However, one difficulty with the development of recombinant factor VIII is achieving production levels in sufficiently high yields.
Various Factor VIII cDNA molecules coding for recombinant factor VIII derivatives have been developed. For example, U.S. Pat. No. 5,661,008 (“the '008 patent”) describes a modified factor VIII derived from a full-length factor VIII cDNA that, when expressed in animal cells, produced high levels of a factor VIII-like protein with factor VIII activity. The protein consisted essentially of two polypeptide chains derived from human factor VIII, the chains having molecular weights of 90 kDa and 80 kDa, respectively. The final active protein is made up of amino acids 1 to 743 and 1638 through 2332 of human factor VIII, the description of which is incorporated by reference herein in its entirety. This polypeptide sequence is commercially known as rFVIII-SQ or REFACTO®. See also, Lind et al., Euro. J. Biochem., 232:19-27 (1995); Sandberg et al., Sem Hematol, 38 (Suppl 4):4-12, 2001.
Other forms of truncated FVIII can also be constructed in which the B-domain is generally deleted. In such embodiments, the amino acids of the heavy chain, consisting essentially of amino acids 1 through 740 of human Factor VIII and having a molecular weight of approximately 90 kD are connected to the amino acids of the light chain, consisting essentially of amino acids 1649 to 2332 of human Factor VIII and having a molecular weight of approximately 80 kD. The heavy and light chains may be connected by a linker peptide of from 2 to 15 amino acids, for example a linker comprising lysine or arginine residues, or alternatively, linked by a metal ion bond.
Affinity chromatography offers a powerful method for protein purification, with the potential to provide exquisite selectivity from contaminating proteins based on the unique interaction between the target protein and ligand immobilized on the resin (Carlsson et al., Affinity chromatography. In: Protein purification: Principles, high resolution methods, and applications. Editors Janson J-C, and Ryden L, New York, Wiley-Liss p 375-442, 1998; Harakas, Biospecific affinity chromatography. In: Protein purification process engineering. Editor Harrison R G, New York, Marcel Dekker p 259-316, 1994). Development of an affinity chromatography step can be difficult if a ligand with suitable affinity or selectivity cannot be identified, or if the elution of the product cannot be achieved without resorting to extreme conditions that may be harmful to the product. While small chemical ligands can be used for affinity separations, their utility has traditionally been restricted to cases where they act as a substrate analog, competitive inhibitor, or co-factor for purification of an enzyme. However, recent work with combinatorial libraries has expanded the repertoire for small molecule ligands (Lowe, Curr Op Chem Biol, 5:248-256, 2001; Morrill, J Chrom B, 774:1-15, 2002). Often, the strength of this binding interaction is moderate to weak (dissociation constants in the millimolar to micromolar range).
At the other extreme, monoclonal antibodies (Mabs) are used as affinity ligands, and often have very high binding affinities (dissociation constants in the nanomolar range) that are difficult to disrupt without extreme pH or high levels of solvents or chaotropes (Goding, Affinity chromatography. In: Monoclonal antibodies: principles and practice. London: Academic Press, p 327-351, 1986). Affinity peptides can be thought of as intermediate between these two cases, as they offer the potential for enormous diversity in chemical properties, and hence selectivity. Further, by using combinatorial methods based on either biological or chemical systems to generate large libraries of unique peptides (Buettner et al., Int J Peptide Protein Res, 47:70-83, 1996; Kaufman et al., Biotechnol Biogen, 77:278-289, 2002; Ladner, Trends Biotechnol, 13:426-430, 1995; Sato et al., Biotechnol Prog 18:182-192, 2002), sequences may be identified with moderate binding affinities that are sufficient to capture the product without undue losses, but which are still capable of eluting the bound protein under mild conditions.
Peptide molecules have also been identified as ligands to be used on affinity chromatography columns. The identification, isolation and synthesis of binding peptide molecules capable of binding factor VIII and/or factor VIII-like polypeptides are disclosed in U.S. Pat. No. 6,197,526. A phage display method is disclosed that is useful for identifying families of polypeptide binding molecules. Using the disclosed method, several binding peptides exhibiting high affinity for factor VIII and factor VIII-like peptides were identified and isolated. The identified peptides were shown to bind REFACTO®. The disclosures of U.S. Pat. No. 6,197,526 are incorporated by reference herein in their entirety.
In Toole et al., Exploration of Structure-Function Relationships in Human Factor VIII by Site-Directed Mutagenesis, Cold Spring Harbor Symposium on Quantitative Biology, 51:543 (1986) it was reported that recombinant FVIII could be purified by a combination of monoclonal antibody or peptide ligand affinity chromatography and ion-exchange chromatography. U.S. Pat. No. 5,470,954 describes a similar process in which FVIII is passed directly from immunoaffinity purification to the ion exchange column. In that document, it is stated that changing the ionic strength or polarity of the eluted polypeptide solution increases the chance that monoclonal antibodies will remain bound to the FVIII polypeptide and co-purify.
U.S. Pat. No. 6,683,159 describes methods for purification of Factor VIII polypeptides by affinity chromatography and ion exchange chromatography. The disclosed method includes the step of diluting the elution solution with a solution comprising higher ionic strength than that of the elution solution, resulting in a diluted Factor VIII solution. In an alternate embodiment, an elution solution is used that has a lower concentration of non-polar agent than that of the desorbing solution. The methods disclosed therein resulted in improved purification without significant yield loss. The disclosure of U.S. Pat. No. 6,683,159 is hereby incorporated by reference in its entirety.